Biotransformation

Dept. of Pharmacology, GMC 1

Amritsar

Drug Absorption

Pharmacological Effect
Blood Pathological Effect

Excretion BIOTRANSFORMATION

Detoxification Activation

Activation
Non-Toxic Metabolite/s Toxic Metabolite/s
De-toxification
Dept. of Pharmacology, GMC 2
Amritsar
 Fate of Drugs
• Drugs are eliminated from the body by the
aid of two processes-
– Metabolism & Excretion
(Biotransformation) (Elimination)
• Elimination is important or else the drug
would stay in the body indefinitely!

Absorption Biotransformation
Elimination
Dept. of Pharmacology, GMC 3
Amritsar

Background
• Drug molecules are processed by
enzymes that have evolved to cope
with endogenous compounds or
those to which we are exposed.

Xenobiotics
Dept. of Pharmacology, GMC 4
Amritsar
 Why Biotransformation?
• Most drugs are excreted by the kidneys
• For renal excretion drugs should:
– have small molecular mass
– be polar in nature
– not be fully ionised at body pH
• Most drugs are complex and do not have
these properties and thus have to be
broken down to simpler products.
Dept. of Pharmacology, GMC 5
Amritsar

• Drugs are lipophilic in nature

• Thus readily pass across biological
barriers –membranes
• Strongly bound to plasma proteins
• This property also stops them from getting
eliminated
• They have to be converted to simpler
hydrophilic compounds so that they are
eliminated and their action is terminated.
Dept. of Pharmacology, GMC 6
Amritsar
 Grease

BIOTRANSFORMATION

SALT
Dept. of Pharmacology, GMC 7
Amritsar

Consequences
• Biotransformation can also result in
bioactivation, which involves the
production of reactive metabolites that are
more toxic, mutagenic, or carcinogenic
than their parent compound(s).
• Drugs may converted to
– less toxic materials
– more toxic materials
– materials with different type of effect or toxicity
Dept. of Pharmacology, GMC 8
Amritsar
 When and How?
• Biotransformations occur between
absorption and elimination from kidneys.
• Drugs administered orally can
biotransform in the intestinal wall.
• Biotransformation reactions can be:
– Phase I reactions
– Phase II reactions

Dept. of Pharmacology, GMC 9

Amritsar

Phase I reactions
• Catabolic in nature
– e.g. Oxidation, Reduction, Hydrolysis
• End-products are chemically more reactive
• Metabolites are usually more polar than
the parent drug.
• Introduce a reactive group – Functionalisation
• The functional group becomes the starting
point for Phase II reaction.
Dept. of Pharmacology, GMC 10
Amritsar
 Phase I
• Cytochrome P450 is the major enzyme
system (Oxidations, reductions, etc.)
• Phase I metabolites may be:
– Inactive
– Equally Active
– More Active
– Toxic
– Activated - “prodrug”
• Polar metabolites may be excreted.
Dept. of Pharmacology, GMC 11
Amritsar

Phase II reactions
• Synthetic processes
• An endogenous substrate is coupled to an
existing (or phase I formed) conjugation
site
• Glucuronic acid, sulphuric acid, acetic
acid, amino acids
• Forms a highly polar conjugate
• Phase II reactions usually occur after
Phase I but can also take place earlier
than Phase I Dept. of Pharmacology,
Amritsar
GMC 12
 Parent Drug Glucuronic acid
or Sulfate
Phase I Glutathione
metabolite others
Dept. of Pharmacology, GMC 13
Amritsar

Where?
• All tissues have the ability to biotransform
drugs
• The liver is ideally placed to intercept
natural ingested xenobiotics and has a
major role in biotransformation
• GIT, Lungs, Skin and Kidneys

Dept. of Pharmacology, GMC 14

Amritsar
 The liver

Hepatocytes
portal smooth bile
venous endoplasmic
blood reticulum
microsomes
contain cytochrome
systemic P450
arterial dependent
blood mixed function oxidases
venous blood

Dept. of Pharmacology, GMC 15

Amritsar

• Certain drugs when administered orally get

absorbed from the intestine and are transported
via the portal system to the liver.
• They are extensively metabolised –First Pass
Effect- limits bioavailability
• Drugs given orally can also get metabolised
– in intestine/
– by intestine wall enzymes
– by microorganisms in lower GIT

Dept. of Pharmacology, GMC 16

Amritsar
 Drug metabolizing enzymes
• Cytochrome P450 (CYP 450) is the major drug
metabolizing enzyme system in the body.
• Comprised of multiple proteins.
• Active site or core of the enzyme system is a
heme protein
• Also known as “mixed function
monooxygenases”
• Liver and gut wall have the greatest
concentration of P450
• Almost all tissues in the body have some P450
(Lungs, Kidney, Skin, Brain, etc)
Dept. of Pharmacology, GMC 17
Amritsar

• Specificity is very low

• Many drugs, chemicals are
metabolised by the CYP 450 enzyme
system.
• Attributes
– Enzyme Induction
– Enzyme Inhibition
– Genetic Polymorphism
Dept. of Pharmacology, GMC 18
Amritsar
 Induction
• Reversible increase in enzyme
concentration
• Resulting from administration of certain
drugs
• Potential to increase rate of the “inducing”
• drug’s breakdown
• Leads to “Pharmacokinetic or Drug
Disposition Tolerance”
• May increase the metabolism of other
drugs taken concurrently
Dept. of Pharmacology, GMC 19
Amritsar

Inhibition
• Some drugs can block P450 enzymes that
metabolise other drugs
• May increase serum concentrations of
second drug
• Can lead to toxicity
• Unlike induction, enzyme inhibition usually
begins with the first dose of the inhibitor.
• Inhibition is maximal when the inhibitor
reaches steady state (four to seven half-
lives). Dept. of Pharmacology, GMC 20
Amritsar
 Cytochrome P450 Enzymes
(mixed function monooxygenases)
• Major drug metabolising enzyme system in the
body.
• Located in the smooth endoplasmic reticulum of
various organs.
• In liver microsomes – Microsomal Enzymes
• liver and gut wall have the greatest
concentration of P450
• Almost all tissues in the body have some P450
(Lungs, Kidney, Skin, Brain, etc)

Dept. of Pharmacology, GMC 21

Amritsar

…CYP 450…
• Substrate specificity is very low
• Varied drugs, chemicals are metabolised
by the CYP 450 enzyme system.
• Powerful oxidising property
• Lipid solubility is important for substrate.
• Attributes
– Enzyme Induction
– Enzyme Inhibition
– Genetic Polymorphism
Dept. of Pharmacology, GMC 22
Amritsar
 Nature
• Haeme protein superfamily of related
enzymes.
• Enzymes differ in Amino acid sequence.
• >74 CYP gene families based on amino
acid sequence are known.
• Three families are responsible for drug
metabolism
• CYP1, CYP2 & CYP3
Dept. of Pharmacology, GMC 23
Amritsar

CYP Isoforms

CYP2E1 CYP1A2
CYP2D6

CYP2A6

CYP2C9

CYP3A4

Dept. of Pharmacology, GMC 24

Amritsar
 Induction
• Results from repeated exposure to certain
xenobiotics including drugs.
• Increased rate of synthesis of enzyme or
reduced rate of breakdown.
• Marked increase in rate of metabolism of
drug-thus decrease in pharmacological
effect.
• Leads to Pharmacokinetic or “Drug
Disposition” Tolerance.
Dept. of Pharmacology, GMC 25
Amritsar

…Induction…
• May also increase the metabolism of other
drugs taken concurrently
• Some drugs are metabolized to reactive
products and may thus exhibit increased
toxicity - Paracetamol.

Dept. of Pharmacology, GMC 26

Amritsar
 Inducers
• Some xenobiotics causing induction:
– Polycyclic hydrocarbons-Epoxide formation
– Smoke- Tobacco
– Charcoal – barbecued food
– Plasticizers
– Dioxin
• Drugs
– Rifampicin
– Ethanol
– Carbamazpine
Dept. of Pharmacology, GMC 27
Amritsar

Inhibition
• Some drugs can block P450 enzyme
isoforms that metabolize other drugs
• May increase serum concentrations of
second drug
• Can lead to toxicity
• Unlike induction, enzyme inhibition usually
begins with the first dose of the inhibitor.
• Inhibition is maximal when the inhibitor
reaches steady state (four to seven half-
lives). Dept. of Pharmacology, GMC 28
Amritsar
 …Inhibition…
• Quinidine inhibits CYP2D6 but is not a
substrate.
• Ketoconazole (antifungal) and known
inhibitor of many enzyme isoforms.
• Clinically important as may lead to drug
interactions.

Dept. of Pharmacology, GMC 29

Amritsar

Other Phase I enzymes

• Not all Phase I reactions are CYP 450
based.
• Other enzymes:
– Alcohol dehydrogenase-Ethanol
– Xanthine oxidase
– Monoamine oxidase-catecholamines
• Reduction reactions
• Hydrolytic reactions
Dept. of Pharmacology, GMC 30
Amritsar
 Reduction reactions-Phase I
• Nitroreductases
• Bacterial reductases
• Aldo-keto reductases
• NADPH-cytochrome-c-reductase

Remove O, add H, decrease

valence
Dept. of Pharmacology, GMC 31
Amritsar

Hydrolysis Reactions-Phase I
• Esterases
• Amidases
• Phosphatases
• Sulphatases
• Epoxide Hydrolase
Add water-expose groups for
Phase II
Dept. of Pharmacology, GMC 32
Amritsar
 Phase II reactions-Enzymes
• Liver, lungs, kidney
• Most of these enzymes are in the cytosol
• Generally occur at a higher rate than Phase I
reactions
• Uridine diphosphate glucuronyl transferase
• Sulphonyl transferase
• Glutathione S-transferase
• N-acetyltransferases + acetyl Co A (NAT +Co A)
• Methyltransferases

Dept. of Pharmacology, GMC 33

Amritsar

…Phase II…
• Glucuronidation
–Major pathway
–Catalyzed by UDP glucuronosyl-
transferase
• Requires UDP co-factor
–Glucuronides are water soluble

Dept. of Pharmacology, GMC 34

Amritsar
 …Phase II…
• Sulfation
• Sulfate conjugation products are
water soluble
• Catalyzed by sulfotransferase
–Transfer of sulfonate

Dept. of Pharmacology, GMC 35

Amritsar

…Phase II…
• Methylation
– Important but minor pathway
– Catalyzed by methyltransferases
• Acetylation
– May or may not be more water soluble
– Increases renal excretion
– Catalyzed by N-acetyltransferase
Dept. of Pharmacology, GMC 36
Amritsar
 …Phase II…
• Amino acid conjugation
–Glycine conjugates
–Taurine conjugates
• Glutathione Conjugation
–Reaction catalyzed by glutathione
s-transferase using glutathione
thiolate
Dept. of Pharmacology, GMC 37
Amritsar

Biotransformation-Conclusion
• Change the xenobiotic to a form that can
be eliminated from the body
• Change the xenobiotic to a less
biologically active form
• Bioactivation to more toxic forms can also
occur
• Catabolic Phase I reactions are carried out
by the CYP450 system
• Synthetic Phase II reactions are carried
out by other enzymes
Dept. of Pharmacology, GMC
Amritsar
38
 Factors Modifying
Biotransformation

Dept. of Pharmacology, GMC 39

Amritsar

Background
• Most patients are treated on similarities
rather than as individuals.
• Same
– Dose
– Problem
– Expected Result
• True for most drugs with a wide margin of
safety
Dept. of Pharmacology, GMC 40
Amritsar
 Examples
• One tablet Paracetamol for fever
• In impaired liver function (alcoholics, hepatitis)
– can cause harm
• Two tablets of Aspirin for toothache
• In patients with renal disease, patients on other
drugs like Warfarin
- can be harmful

Dept. of Pharmacology, GMC 41

Amritsar

Sources of variation
• Age •Species difference
• Gender •Pregnancy
• Environmental factors
• Diet
• Disease
• Drug-drug interactions
– Enzyme induction
– Enzyme inhibition
• Genetic factors

Dept. of Pharmacology, GMC 42

Amritsar
 Age
• Extremes of age are associated with
disturbances in metabolism of drugs.
• In paediatric age group
– Premature infants, neonates, children and
adolescents cannot be treated like small
adults.
• All these groups have special metabolic
parameters.
• Foetus: CYP3A Sub-family only poor
metabolism.
• Neonates virtually no Phase-2 enzymes
Dept. of Pharmacology, GMC 43
Amritsar

…Age…
• Hepatic biotransformation and enzyme
activity is reduced in the early neonatal
stages.
• There is decreased biotransformation of
drugs and increased plasma levels and
prolonged half life.
• Less developed excretory mechanisms.
• Malnutrition in children can impair
metabolism.
Dept. of Pharmacology, GMC 44
Amritsar
 Gray-baby syndrome.
• Chloramphenicol toxicity leading to
Inadequate glucuronidation due to
diminished glucuronyl transferase activity
– Immature kidney exhibits inadequate renal
excretion of unconjugated drug and
glucuronide conjugate.
– Elimination half life 26 hours in neonates
– 4 hours in older children
N o rm a l

E xc r e t i o n

N e o na t e s

0
Dept.
10
of Pharmacology,
20
GMC 30
45
Amritsar

Elderly Patients
• In patients > 65 years complex
pharmacokinetic changes occur.
• Decrease in liver size and liver blood flow
• Activity of phase I pathways is reduced
thus drugs predominantly metabolised by
this path may show an exaggerated
response.
– eg. Diazepam as sedative
• Irregular eating habits (PCM) and vitamin
deficiencies are associated with impaired 46
Dept. of Pharmacology, GMC
metabolism Amritsar
 …Elderly…
• Diminished enzyme induction
• Drug drug interactions are more common
– Larger number of drugs being prescribed.
– Both induction and inhibition can result.
• Renal excretion of drugs and metabolites
is impaired

Dept. of Pharmacology, GMC 47

Amritsar

Gender
• Seen and documented in experimental animals.
• Usually associated with sex hormones.
• Menstrual cycle affects metabolism
• In humans a difference in metabolism of a few drugs has
been reported:
– Alcohol
– Benzodiazepines
– Some antiinflammatory drugs
– Propranolol oxidation M>F.
– Morphine
• N-Demethylation of erythromycin F>M.

Dept. of Pharmacology, GMC 48

Amritsar
 Pregnancy
• In pregnancy there is a concern for foetus
• Placenta high in CYP1A family if smoker.
• Consequences to foetus or neonate:
teratogenicity, carcinogenicity,
hepatotoxicity
• Can have profound induction in
pregnancy.
e.g., may have to increase
anticonvulsants.
Dept. of Pharmacology, GMC 49
Amritsar

Environmental factors
• Cigarette smoke leads to enzyme
induction and increases the breakdown of
drugs.
• Exposure to industrial chemicals,
pollutants also alters metabolism.
• Clinical outcome:
– Increase dose in smokers
– Drugs with narrow safety margins should be
given carefully.
Dept. of Pharmacology, GMC 50
Amritsar
 …Disease…
• Cardiac disease leads to decreased blood
flow to liver and delayed metabolism.
• Pulmonary disease may impair
metabolism of certain drugs.
• Thyroid disorders may lead to fast
metabolism-hyperthyroidism or vice versa.

Dept. of Pharmacology, GMC 51

Amritsar

Drug-drug interactions
• When two or more drugs are co-
administered
– Enzyme inducers may lead to increased
metabolism of other drugs.
– There is impaired elimination of slowly
metabolized drug and thus prolonged effect
and toxicity.

Dept. of Pharmacology, GMC 52

Amritsar
 Enzyme induction

Oral Contraceptives CYP3A4 Inactivated

Steroids Excreted
Induction

Rifampicin
Contraceptive
failure
Dept. of Pharmacology, GMC 53
Amritsar

Enzyme induction

CYP2E1
Paracetamol Toxic
Metabolite
Induction

Toxicity Ethanol

Dept. of Pharmacology, GMC 54

Amritsar
Enzyme induction/ Herbal Drug

CYP3A4
Cyclosporin Metabolite

Induction
Impaired
Immune
St. Johns Wort
response
Rejection
Dept. of Pharmacology, GMC 55
Amritsar

Enzyme inhibition

CYP3A1
CYP2B2
Warfarin Inactivation

Inhibition

Impaired Chloramphenicol
control of
Bleeding
Dept. of Pharmacology, GMC 56
Amritsar
 Enzyme Inhibition

CYP3A4
Terfenadine Active
antihistamine
Inhibition
Erythromycin
Arrhythmias
Ketoconazole
Grapefruit juice
Dept. of Pharmacology, GMC 57
Amritsar

Genetic factors
• Pharmacogenetics as a discipline
• Explains why one patient's response
to drug therapy is different from
another patient's when both are
being treated with the same drug for
the same problem
• Provides an understanding of the
outcomes of therapy.
Dept. of Pharmacology, GMC 58
Amritsar
 Pharmaco-genetics/ genomics
• Pharmacogenetics is to use a
patient's genetic profile to optimize
drug therapy and minimize drug
toxicity
• Pharmacogenomics: identifying
innovative drug targets and
accounting for the effect that DNA-
sequence variations have on a drug's
effectiveness.
• Pharmacogenomics may ultimately
result in the development of new
medications Dept.
forof Pharmacology,
unmetGMCmedical needs59
Amritsar

• Pharmacogenetics studies gene

variations, which can exist either as
– rare defects (eg, mutations seen in
diseases like cystic fibrosis) or
– as polymorphisms-a genetic variation
that occurs in 1% or more of humans.
• Many of the known polymorphisms
involve cytochrome P-450 (CYP450)
isoenzymes.
Dept. of Pharmacology, GMC 60
Amritsar
 Examples
Drug Genetic Variation Result
Codeine Defective CYP2D6 Decreased
gene; conversion to
morphine cannot analgesia
occur

Phenytoin Defective CYP2C9 Ataxia

gene, resulting in
overdosage Confusion

Defective CYP2C9 Bleeding

Warfarin gene; decreased
warfarin clearance
Dept. of Pharmacology, GMC 61
Amritsar

• Ethnicity has a role in determining how

well a patient metabolizes drugs
• Categorised as: Poor/ intermediate/
extensive and ultrarapid metbolisers.
• The incidence of toxicity or decreased
efficacy depends on how the specific
variant of the gene affects an enzyme,
causing
– poor metabolism (resulting in toxicity) or
– extensive metabolism (resulting in decreased
efficacy) of the drug.
Dept. of Pharmacology, GMC 62
Amritsar
 Isoniazid
• Slow acetylator phenotype leads to
accumulation of the drug and enhances
toxicity manifested as peripheral
neuropathy
• Fast acetylators tend to metabolise the
drug rapidly leading to low therapeutic
levels and hepatotoxicty.

Dept. of Pharmacology, GMC 63

Amritsar

Species Differences

• Inter- and intraspecies variation.

• Cats form sulfates (lack UDP-GT) but
Pigs form glucuronides (lack of AST).
• Animal models for predicting metabolism.
• Humans have ONE CYP2D isoform
(CYP2D6).
• Rats have SIX CYP2D isoforms.

Dept. of Pharmacology, GMC 64

Amritsar